DEMETHYLATION OF THE PESTICIDE METHOXYCHLOR IN LIVER AND INTESTINE FROM UNTREATED, METHOXYCHLOR-TREATED, AND 3-METHYLCHOLANTHRENE-TREATED CHANNEL CATFISH (ICTALURUS PUNCTATUS): EVIDENCE FOR ROLES OF CYP1 AND CYP3A FAMILY ISOZYMES

Abstract

Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 ± 1.3 μM (mean ± S.D., n = 4), and Vmax was 131 ± 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 ± 0.8 μM and Vmax 99 ± 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 ± 1.1 μM and Vmax of 246 ± 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 ± 4 to 15 ± 6 pmol/min/mg (mean ± S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 ± 22 pmol/min/mg. Ketoconazole, clotrimazole, and α-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas α-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] is an organochlorine pesticide that was developed as a substitute for DDT [1,1,1-trichloro-2,2-bis(chlorophenyl)ethane]. In contrast to DDT, methoxychlor exhibits generally low overt toxicity and bioaccumulation in mammals. Methoxychlor has been used as a pesticide on pets, livestock, crops, gardens, and in animal feed (Palanza et al., 2001; Magliulo et al., 2002). The use of methoxychlor in the United States legally ended in 2004 when the chemical was denied reregistration by the U.S. Environmental Protection Agency; however, widespread use over the past half-century has produced considerable environmental contamination and exposure (Li and Kupfer, 1998). A recent study of house dust samples from 120 homes in Cape Cod, Massachusetts found a median methoxychlor concentration of 0.24 μg/g (Rudel et al., 2003). Tissues of several arctic wildlife species, including snow crab and narwhal, had detectable residues of methoxychlor, indicating global distribution of the pesticide (Vorkamp et al., 2004).

As is the case with DDT, methoxychlor has estrogenic metabolites and produces endocrine disruption in fish (Larkin et al., 2003; Borgert et al., 2004). Evidence has been presented that suggests that the demethylated metabolites of methoxychlor, 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (OH-MXC) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (HPTE), are more potent estrogens in channel catfish than either o,p′-DDT or 1,1-dichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethene (Nimrod and Benson, 1997). Likewise, OH-MXC has been shown to be 16 to 43 times more potent than the parent methoxychlor for binding to hepatic estrogen receptors in channel catfish (Schlenk et al., 1998). Experiments with expressed human and rat estrogen receptors α and β indicated that both OH-MXC and HPTE acted as agonists at estrogen receptor α but as antagonists at estrogen receptor β, as well as at androgen receptors (Gaido et al., 1999, 2000). Thus, the biotransformation of methoxychlor to demethylated metabolites is an important toxication step.

Cytochrome P450 (P450)-catalyzed O-demethylation of methoxychlor results in the sequential formation of OH-MXC and HPTE (Fig. 1). In human liver microsomes, several P450 isoforms, but predominantly CYP2C9 and CYP1A2, catalyze demethylation of methoxychlor to OH-MXC (Hu and Kupfer, 2002; Hu et al., 2004; Hazai and Kupfer, 2005). Demethylation of OH-MXC to form HPTE was catalyzed by human CYP1A2, CYP2C8, CYP2C19, CYP2D6, and to a lesser extent CYP3A4 (Hu and Kupfer, 2002). Although there have been several studies of the adverse effects of methoxychlor in various fish species, little is known of the biotransformation of methoxychlor in fish. Studies by Schlenk et al. (1997), with the environmentally relevant channel catfish model, have examined the influence of methoxychlor pretreatment and P450 induction by β-naphthoflavone on methoxychlor biotransformation in catfish hepatic microsomes. These studies showed that high-dose exposures to methoxychlor (2 × 254 mg/kg i.p.) or this dose of methoxychlor in combination with β-naphthoflavone (50 mg/kg i.p.) both reduced the rates of methoxychlor demethylation to OH-MXC relative to untreated controls or fish receiving only β-naphthoflavone. β-Naphthoflavone treatment alone did not increase the rate of methoxychlor demethylation. The effects of lower, more environmentally relevant doses of methoxychlor or the effects of other aryl hydrocarbon receptor inducers were not examined.

Because Hu and Kupfer (2002) showed that human CYP1A2 readily metabolized methoxychlor, it was somewhat surprising that the CYP1 family inducer β-naphthoflavone did not increase methoxychlor metabolism in catfish (Schlenk et al., 1997). However, it has been shown that residues of β-naphthoflavone can inhibit CYP1A (James et al., 1997); therefore, it was of interest to examine the effects of another polycyclic aromatic inducing agent, 3-methylcholanthrene (3-MC). Gaining a better understanding of the P450-dependent metabolism of methoxychlor to estrogenic metabolites would help establish which species were more susceptible to the pesticide and which exposures to other chemicals could induce or inhibit the production of estrogenic metabolites. As well as CYP1 family inducers, such as polyaromatic hydrocarbons and polychlorinated biphenyls, fish may be exposed to a variety of agricultural chemicals, some of which interact with P450 and may affect methoxychlor biotransformation. Imidazole and triazole-containing agrochemicals such as prochloraz, propiconazole, and thiobendazole interact with P450 acutely to inhibit monooxygenation (Snegaroff and Bach, 1989), similarly to the anti-fungal drugs ketoconazole and clotrimazole, although there is evidence that chronic exposure to azoles induces P450 in fish (Egaas et al., 1999; Hegelund et al., 2004). This research aims to create a better understanding of the demethylation of methoxychlor in channel catfish and subsequently the factors creating the endocrine-disrupting toxicity of methoxychlor.

Materials and Methods

Chemicals. Methoxychlor used to treat catfish and in the preparation of metabolite standards was obtained from ICN (Aurora, OH) and was >99% pure. NADPH, clotrimazole, 3-MC, and [14C]methoxychlor (9.6 mCi/mmol) were obtained from Sigma Chemical Co. (St. Louis, MO). Before use in assays, the [14C]methoxychlor was purified to >99.9% by normal-phase silica gel thin-layer chromatography on LK5DF plates (Whatman, Florham Park, NJ) developed in diethyl ether/n-heptane, 1:1 by volume. 7-Ethoxyresorufin was synthesized by ethylation of resorufin, and both 7-ethoxyresorufin and resorufin were purified by thin-layer chromatography on preparative silica gel plates developed in chloroform/toluene/ethyl acetate/acetone, 3:3:2:1 by volume (Prough et al., 1978). Ketoconazole was obtained from Janssen Pharmaceutica (Piscataway, NJ), and α-naphthoflavone was obtained from Aldrich (Milwaukee, WI). All the chemicals were at least 98% pure.

Preparation of Metabolite Standards. OH-MXC and HPTE were prepared by boron tribromide-catalyzed demethylation of methoxychlor using the method of Hu and Kupfer (2002). The OH-MXC and HPTE products were separated by silica gel column chromatography and further purified by recrystallization from diethyl ether/n-heptane. Their identities were established by melting point and proton NMR (Kapoor et al., 1970). OH-MXC had a melting point of 109°C, and 1H NMR in CDCl3 showed peaks at δ3.79 (s, 3H, O-CH3), 4.84 (s, 1H, OH), 4.95 (s, 1H, CHCl3), 6.78 to 6.89 (m, 4H, aromatic), and 7.46 to 7.53 (m, 4H, aromatic). HPTE had a melting point of 204°C, and 1H NMR in D6-dimethyl sulfoxide showed peaks at δ5.12 (s, 1H, CHCl3), 6.71 to 6.73 (m, 4H, aromatic), 7.45 to 7.47 (m, 4H, aromatic), and 9.46 (s, 2H, OH). Reverse-phase high-performance liquid chromatography with UV detection at 245 nm was used to verify the purity of each methoxychlor metabolite. The column was a 25 cm × 4.6 mm, 5-μm C18 Discovery column fitted with a 2 cm × 4.6 mm, 5-μm C18 Discovery guard column (Supelco, Bellefonte, PA). The mobile phase was isocratic acetonitrile/water (70:30) at 1 ml/min. Both metabolite standards were shown by high-performance liquid chromatography analysis to be >99% pure.

Animals. Male and female adult channel catfish weighing between 1100 g and 2700 g (average 1675 g) were used in these studies. The catfish were fed a purified diet as described previously (James et al., 1997). Four catfish were used in each treatment group. These were untreated (control), treated daily for 6 days with 2 mg/kg methoxychlor in corn oil by i.p. injection before sacrifice on day 7 (methoxychlor), or treated with a single i.p. injection of 10 mg/kg 3-MC in corn oil 3 to 5 days before sacrifice (3-MC). The catfish were sacrificed by stunning with a blow to the head, followed by severing the spinal cord behind the head. This procedure was approved by the University of Florida Institutional Animal Care and Use Committee. The liver was removed and, after excision of the gall bladder, placed in ice-cold buffer 1 (0.15 M KCl, 0.05 M potassium phosphate, pH 7.4, and 0.2 mM phenylmethylsulfonyl fluoride). After rinsing three times in this buffer, the liver was minced and homogenized in 4 volumes of buffer 1. The whole intestine was removed, rinsed with ice-cold buffer 2 (0.25 M sucrose, 5 mM EDTA, 0.05 M Tris-Cl, pH 7.4, and 0.2 mM phenylmethylsulfonyl fluoride) to remove particles, and sliced open longitudinally. The intestinal mucosa was scraped with a scalpel into 10 ml of buffer 2, and mucosal cells were sedimented by centrifugation at 2000g. The mucosal cells were homogenized in 4 volumes of buffer 2. Washed hepatic and intestinal microsomes were prepared from all the catfish as described previously and resuspended in a volume of buffer containing 0.25 M sucrose, 0.01 M Hepes buffer, pH 7.4, 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride that was equal to the original wet weight of the liver or intestinal mucosa (James et al., 1997). The washed microsomes were stored under nitrogen at -80°C in 0.5-ml aliquots until use. Studies established that monooxygenase activity was stable for at least 6 months under these storage conditions.

Methoxychlor Monooxygenation Assay. A radiochemical method was developed with extraction and thin-layer chromatography analysis of metabolites. The optimal conditions for solvent extraction of methoxychlor and metabolites and for the incubation time and protein concentrations for linear product formation in hepatic and intestinal microsomes were determined experimentally. Assay tubes contained, in a final volume of 0.5 ml, [14C]methoxychlor (varying concentrations), 0.1 M Hepes, pH 7.6, 50 mM MgCl2, liver microsomes (0.2 mg of protein) or intestinal microsomes (0.3 mg of protein), and 2 mM NADPH. In all the experiments, the methoxychlor was added to empty tubes in ethanol solution (0.005 ml), and in most studies, the ethanol was removed under nitrogen before addition of other components, except NADPH. The tubes were placed in a 35°C shaking water bath for 2 min, and then NADPH was added. After 15 min, the reaction was stopped by the addition of 0.5 ml of ice-cold water and 2 ml of water-saturated ethyl acetate. Each tube was vortex-mixed, and the phases were separated by centrifugation. The ethyl acetate layer was transferred to a clean tube. Two more extractions were performed with 2 ml of ethyl acetate. The pooled ethyl acetate fractions were dried by addition of anhydrous sodium sulfate. The dry ethyl acetate was transferred to a clean tube; the solvent was evaporated under nitrogen; and the residue of methoxychlor and metabolites was dissolved in 0.2 ml of ethanol. Aliquots of each sample, 20 to 50 μl, were applied to LK6DF normal-phase silica gel thin-layer chromatography plates with fluorescence indicator (Whatman), along with standards for methoxychlor, OH-MXC, and HPTE. The plates were developed in n-heptane/diethyl ether, 1:1 by volume. The standards were visualized with UV light. The [14C]methoxychlor and metabolites were detected and quantitated by overnight electronic autoradiography with a Packard Instant Imager (Meriden, CT). Rates of formation of OH-MXC and HPTE were calculated from the percentage of each metabolite produced and the known concentration of methoxychlor present in assay tubes.

Km and Vmax Determination.Km, Vmax, and clearance values for formation of OH-MXC by liver microsomes were calculated from the rates found at several concentrations of methoxychlor and the Michaelis-Menten equation using Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA). In studies where 1% v/v ethanol remained in the assay tubes, final methoxychlor concentrations of 5, 10, 15, 50, and 100 μM were used to determine Km values. In experiments where the ethanol was removed before addition of other assay components, final methoxychlor concentrations of 2, 5, 10, 15, and 25 μM were used.

Solvent Inhibition Studies. Ethanol and acetone were used to prepare solutions of methoxychlor and several chemical P450 inhibitors. The effects of these solvents on the demethylation of methoxychlor were examined. Solvents were added (1% ethanol v/v, 2% ethanol v/v, and 1% ethanol v/v with 1% acetone v/v) to assay tubes after addition of all the other components except NADPH, and rates of formation of OH-MXC were compared with rates in tubes without solvent.

Chemical Inhibition Studies. The effects of ketoconazole, α-naphthoflavone, and clotrimazole on methoxychlor demethylation were examined. The inhibitors were dissolved in ethanol and added to the assay at the same time as 25 μM methoxychlor. The solvent was evaporated under nitrogen so that no ethanol remained in the tube before addition of other assay components. Five different concentrations of each inhibitor were used. From the inhibition of the monodemethylation of methoxychlor, an IC50 value was calculated for each inhibitor by plotting the log concentration of inhibitor versus the fraction of activity remaining. Where possible, the effects on HPTE formation were also examined.

Monoclonal Antibody Inhibition Studies. Monoclonal antibody to scup CYP1A (MAb-1-12-3) was prepared as described previously (Stegeman et al., 1985; Park et al., 1986). Monoclonal antibody to egg lysozyme (MAb Hy-Hel-9) in ascites fluid was provided by K. Krausz (National Institutes of Health, Bethesda, MD) and used as a control (Krausz et al., 2001). Ascites fluid containing monoclonal antibody to scup CYP1A was incubated on ice with microsomes from 3-MC-induced channel catfish in the ratio 20 or 40 μg of antibody/pmol total measured P450 for 15 min. The total P450 content of microsomes was measured spectrally as described previously (James et al., 1997). Ascites fluid containing the MAb Hy-Hel-9 was similarly incubated with microsomes from 3-MC-induced channel catfish in the ratio 1 μl/pmol total P450. The mixture of microsomes and monoclonal antibody was added to assay tubes prepared as described above, containing 25 μM methoxychlor. The tubes were placed in a shaking water bath at 35°C for 3 min before addition of NADPH.

Ethoxyresorufin O-Deethylation Activity. Ethoxyresorufin O-deethylation (EROD) activity was measured to establish that treatment of catfish with 3-MC induced CYP1A, as well as a positive control for the effects of the monoclonal antibody to scup CYP1A on methoxychlor demethylation. A fluorescence assay was used as described previously (James et al., 1997). Assay tubes contained 2.5 μM ethoxyresorufin, 0.1 M Hepes buffer, pH 7.6, 0.25 mg/ml of microsomal protein (controls) or 0.005 mg/ml of microsomal protein (3-MC-treated), and 2 mM NADPH in a total volume of 0.5 ml and were incubated at 35°C for 5 min (controls) or 2 min (3-MC-treated). To study the effect on EROD activity of the monoclonal antibody to scup CYP1A, microsomes from 3-MC-induced fish were preincubated for 15 min on ice with 0.1 M Hepes buffer, pH 7.6, and MAb-1-12-3 in the ratio of 20 or 40 μg of MAb/pmol total measured total P450 before use in assays. Three sets of controls were used. One set contained microsomes from 3-MC-induced fish that were preincubated on ice with 0.1 M Hepes buffer, pH 7.6, and MAb Hy-Hel-9 in the ratio of 1 μl/pmol P450; a second contained microsomes from 3-MC-induced fish that were preincubated on ice for 15 min with 0.1 M Hepes buffer, pH 7.6, and a volume of water equal to that of the ascites fluid; and a third set was not preincubated.

Western Blot Analysis of Liver CYP1A Protein. The induction status of 3-MC-treated fish and the amount of CYP1A in liver microsomes from control catfish were examined by immunoblot analysis. Monoclonal antibody to scup CYP1A (MAb-1-12-3) was used (Park et al., 1986). Hepatic microsomes from control (40 μg of protein) and 3-MC-treated (5 μg of protein) fish, as well as unstained and prestained molecular weight standards in the range 14,400 to 97,000, were separated on a 4% stacking and 8.5% resolving SDS-polyacrylamide mini-gel as described by Laemmli (1970). The electrophoretically resolved proteins were transferred to a nitrocellulose membrane, blocked with nonfat milk, and incubated with mouse anti-scup CYP1A (1:10,000) at 4°C overnight. The membrane was washed, incubated with 1:1000 sheep anti-mouse IgG conjugated with horseradish peroxidase, rewashed, and developed with Super Signal (Pierce, Rockford, IL) for 5 min. Membranes with control liver protein were exposed to film for 15 min, and membranes with 3-MC-treated protein were exposed to film for 15 s. The intensities of the bands were compared with varying known amounts of rat CYP1A as reference standards (BD Gentest, Woburn, MA).

Results

Assay Development in Liver Microsomes. Incubations to investigate the demethylation of methoxychlor were initially performed with a procedure similar to those used by other investigators (Schlenk et al., 1997; Stresser and Kupfer, 1998; Hu and Kupfer, 2002). In the published procedures and our initial studies, [14C]methoxychlor was dissolved in ethanol and added to the reaction mixture for a final concentration of 1% ethanol in the assay tubes. The metabolites were separated by thin-layer chromatography. Methoxychlor displayed an RF of 0.63, OH-MXC had an RF of 0.33, and HPTE had an RF of 0.17. No other products were identified, although a small amount of radioactivity remained on the preadsorbent band of the silica gel plates. The time course of formation of metabolites was investigated by incubating tubes for 5, 10, 15, 20, 30, or 45 min. Production of OH-MXC by channel catfish liver microsomes was linear up to 30 min. After a lag phase of 10 min, HPTE production was linear for at least 45 min. The effect of varying protein concentration was investigated by conducting incubations for 15 min. The formation of OH-MXC was linear with up to 0.35 mg of protein/tube, and HPTE formation was linear up to 0.6 mg of protein/tube.

Solvent Inhibition. In preparing to conduct studies with monooxygenase inhibitors, the possibility of solvent inhibition of methoxychlor metabolism was investigated. It was found that 1% ethanol in incubation tubes inhibited formation of OH-MXC in catfish liver microsomes by 44%. Addition of ethanol to 2% of the assay volume or addition of 1% ethanol and 1% acetone inhibited monooxygenation by 71% and 64%, respectively. To avoid solvent inhibition, all the solvents were removed from assay tubes by evaporation under nitrogen before adding the other components of the reaction mixture.

Kinetics of Methoxychlor Monodemethylation in Liver and Effect of Treatment. The kinetic constants Km and Vmax for OH-MXC formation in liver microsomes from control and methoxychlor-pretreated catfish were obtained in the presence of 1% ethanol before the effect of ethanol on activity was recognized. Rates of OH-MXC formation fit the Michaelis-Menten equation in the range 2.5 to 100 μM methoxychlor in most fish, although in two fish, rates started to decrease at methoxychlor concentrations >50 μM (data not shown). For those fish that showed lower rates at 100 μM methoxychlor, this point was omitted from the calculations of Km and Vmax values. After discovery of the ethanol inhibition, Km and Vmax values for methoxychlor demethylation were obtained in the same liver samples without using ethanol in the procedure and using a range of methoxychlor concentrations (2.5-25 μM) that bracketed the Km and fit the Michaelis-Menten equation. Results for all the kinetic studies are listed in Table 1. Pairwise comparisons of Km and Vmax values in the same control fish or fish pretreated with methoxychlor revealed significantly lower Km values in the absence of ethanol (p < 0.005) than those obtained with ethanol, whereas Vmax values were significantly higher (p < 0.05) when ethanol was removed from the procedure.

Kinetic data for the formation of OH-MXC in hepatic microsomes from control, methoxychlor-treated, and 3-MC-treated channel catfish Values shown are mean ± S.D., n = 4 fish per treatment group. Methoxychlor-treated fish were injected i.p. with 2 mg/kg methoxychlor for 6 days before sacrifice on day 7. 3-MC-treated fish were injected i.p. with 10 mg/kg 3-MC 3 to 5 days before sacrifice.

Table 1 also shows the influence of treatment with methoxychlor or 3-MC on methoxychlor demethylation kinetics without the influence of ethanol. Figure 2 shows Michaelis-Menten graphs for representative control, methoxychlor-treated, and 3-MC-treated fish. There were no significant differences in the Km and Vmax values for control and methoxychlor-treated fish (Table 1); however, there was a large spread in Vmax values in both groups. Treatment of catfish with 3-MC significantly (p < 0.05) altered the kinetics of OH-MXC production compared with control catfish, with increases in both Km and Vmax. The 3-MC-treated fish showed less variability for Vmax than control or methoxychlor-treated catfish.

The 3-MC-treated fish were shown to have induced CYP1A in hepatic microsomes. The EROD activity (mean ± S.D., n = 4) for control fish was 19 ± 5 pmol/min/mg protein, whereas 3-MC-treated fish had mean activity of 1078 ± 268 pmol/min/mg protein, a 58-fold increase (p < 0.001). Western blot analysis showed an increase of CYP1A protein, as seen in Fig. 3, A and B, for control and 3-MC-treated fish, respectively. Control fish had an average of 1.7 ± 0.2 units CYP1A/mg protein, whereas 3-MC-treated fish had an average of 1040 ± 225 units CYP1A/mg protein, where one unit was the signal density produced by 1 pmol rat CYP1A. The increase in CYP1A protein was significant (p < 0.001).

Formation of HPTE in Liver. The secondary metabolite, HPTE, was formed in incubations with channel catfish liver microsomes. Activity data from the three treatment groups of fish were compared for incubations with 25 μM [14C]methoxychlor (Table 2). HPTE formation in most fish treated with methoxychlor was below the detection limits (<1 pmol/min/mg protein). Fish treated with 3-MC formed HPTE at significantly higher rates (p < 0.05) than the control. Under the assay conditions used, which were optimized for OH-MXC formation, the amounts of HPTE formed were significantly lower than OH-MXC in all the groups of fish (p < 0.01).

Rates of formation of OH-MXC and HPTE in hepatic microsomes and OH-MXC in intestinal microsomes from control, methoxychlor-treated, and 3-MC-treated channel catfish Values shown are pmol/min/mg protein and are mean ± S.D., n = 4 fish per treatment group. Fish were treated as described in Table 1.

Rates of formation of OH-MXC at differing methoxychlor concentrations with hepatic microsomes from representative control (▪), methoxychlor-treated (▴), and 3-MC-treated (•) channel catfish. Results shown are means of replicate samples from individual fish. The curves show the fit of the data to the Michaelis-Menten equation.

Intestinal Demethylation of Methoxychlor. The monooxygenation of methoxychlor in intestinal microsomes was examined in samples from control, methoxychlor, and 3-MC-treated fish using 15 μM [14C]methoxychlor. This concentration was selected as being low, yet saturating in liver microsomes from control fish (Fig. 2). Fish treated with 3-MC had significantly higher intestinal activity (p < 0.05) than the control fish (Table 2). The intestinal activity of the methoxychlor-treated group was significantly lower than the control (p < 0.01) (Table 2). Although HPTE was formed in some intestinal samples (data not shown), the amounts were generally too small for accurate quantitation under the incubation conditions used. Kinetic studies were not performed with intestinal microsomes because of the relatively small amount of intestinal mucosa obtained and the lower overall activity in intestines.

Inhibition of Methoxychlor Monooxygenation. Ketoconazole, clotrimazole, and α-naphthoflavone all inhibited the formation of OH-MXC in hepatic microsomes from each of the catfish treatment groups, but with different inhibitory potencies. The mean IC50 values are summarized in Table 3. There was no significant treatment difference (p > 0.05) in the IC50 values for ketoconazole or clotrimazole. Clotrimazole was slightly more potent than ketoconazole. α-Naphthoflavone was less potent than the other two inhibitors. Solubility for α-naphthoflavone was a limiting factor for accurate measurements in incubations containing >100 μM; thus, it was not possible to show more than about 70% inhibition with this compound (data not shown). There was no significant difference (p > 0.05) in the α-naphthoflavone IC50 between control and methoxychlor-treated catfish, but α-naphthoflavone was a more potent inhibitor (p < 0.05) in 3-MC-treated catfish (Table 3). Whereas increasing concentrations of α-naphthoflavone decreased the amount of OH-MXC produced by hepatic microsomes from 3-MC-treated catfish, the amount of HPTE produced increased. In a representative 3-MC-treated fish, HPTE formation was 14 pmol/min/mg protein in assays with no added modifier, increased to 22 pmol/min/mg protein in the presence of 25 μM α-naphthoflavone, and remained elevated at higher concentrations up to 100 μM, after which HPTE formation declined (Fig. 4A). Similar results were found with hepatic microsomes from the other 3-MC-treated fish. Because α-naphthoflavone inhibited formation of OH-MXC, the rate of HPTE formation expressed as a percentage of OH-MXC formation increased with increasing amounts of α-naphthoflavone (Fig. 4B).

Although 3-MC treatment increased methoxychlor monooxygenation, a monoclonal antibody to scup CYP1A did not inhibit the rate of formation of OH-MXC in liver microsomes from 3-MC-induced catfish compared with control incubations conducted in the presence of antilysozyme ascites fluid or incubations with no added antibody. Indeed, there was a slight increase in activity in the presence of MAb-1-12-3 (Fig. 5A). Studies conducted to determine whether this monoclonal antibody would inhibit catfish CYP1A showed that MAb-1-12-3 did inhibit EROD activity in hepatic microsomes from 3-MC-induced catfish. Tubes containing 20 μg of antibody/pmol total P450 showed a 20% inhibition of EROD activity, and tubes containing 40 μg of antibody/pmol total P450 were 42% inhibited (Fig. 5B).

Effect of increasing concentrations of α-naphthoflavone on the demethylation of methoxychlor (25 μM) in hepatic microsomes from 3-MC-treated catfish. A, rates of formation of OH-MXC (•) and HPTE (▴). B, HPTE formation expressed as a percentage of the OH-MXC produced. Results shown are the means of replicate samples from an individual fish. Similar results were observed with other fish.

Discussion

This study showed that ethanol inhibited methoxychlor demethylation in catfish liver microsomes (Table 1). The presence of 1% ethanol in assay tubes approximately doubled the Km for methoxychlor in control and methoxychlor-pretreated fish and, on a fish-by-fish basis, decreased Vmax. In the earlier study of methoxychlor metabolism in catfish liver microsomes, Schlenk et al. (1997) used 1% ethanol to add methoxychlor, making it difficult to compare our results with the earlier study. The ethanol effect on Vmax and Km that we observed suggests a mixed-type inhibition by ethanol; however, detailed studies of the kinetics of ethanol inhibition were not carried out. The competitive portion of the inhibitory interaction may indicate the involvement of a CYP2E1-like isoform, which metabolizes ethanol (Gonzalez, 2005), in methoxychlor demethylation in the catfish.

The finding that Km values in untreated catfish were in the low micromolar range suggested that environmentally exposed catfish would rapidly metabolize methoxychlor to active estrogenic metabolites in the liver. Demethylation rates catalyzed by intestinal microsomes were lower than by liver microsomes, and because the intestine is a rather small organ in catfish, this suggests limited intestinal metabolism from dietary exposure to methoxychlor. We did not examine kinetics in the intestinal microsomes because of their lower methoxychlor demethylase activities and the relatively small amount of intestinal mucosa in catfish.

Considerably more OH-MXC than HPTE was formed by catfish liver microsomes in the 15-min incubation time used in most of these studies (Table 2). Human liver microsomes incubated under similar conditions (25 μM methoxychlor for 10 min) likewise formed less HPTE than OH-MXC, with amounts of HPTE ranging from nondetectable to about half the amount of OH-MXC formation (Stresser and Kupfer, 1998). As with human and rat liver microsomes, the relative production of HPTE by catfish liver microsomes increased with longer incubation times, concurrent with increased formation of OH-MXC, the precursor of HPTE.

Effect of a monoclonal antibody to fish (scup) CYP1A (MAb-1-12-3) on OH-MXC formation (A) and EROD activity (B) in hepatic microsomes from a 3-MC-treated catfish. Similar results were observed with other fish.

Treatment of catfish with 2 mg/kg methoxychlor for 6 days did not change the hepatic Km or Vmax values for OH-MXC formation, suggesting that subchronic exposure to low concentrations of methoxychlor will not change its initial biotransformation in the liver. In intestine, however, this treatment reduced demethylation activity to half that of untreated controls. The reduction in methoxychlor demethylation suggests that methoxychlor, or a metabolite, down-regulates or inhibits one or more of the P450 isoforms responsible for OH-MXC formation in the catfish intestine. This finding differs from the reported effects of methoxychlor in rats. Treatment of rats with high doses of methoxychlor reduced the formation of OH-MXC but increased the formation of HPTE and ring-hydroxylated metabolites by liver microsomes, suggesting no effect on the initial formation of OH-MXC, but rather more rapid further metabolism in the methoxychlor-treated rats (Li et al., 1995). In the rat, methoxychlor, OH-MXC, HPTE, and other metabolites activated the constitutive androstane receptor system and induced CYP2B and CYP3A, consistent with the observed increased metabolism of methoxychlor and OH-MXC (Blizard et al., 2001). There was also evidence in rats that methoxychlor induced synthesis of CYP3A through the pregnane X receptor (PXR) (Mikamo et al., 2003). Although the effects of methoxychlor or its metabolites on the isoform composition of catfish liver or intestine are not known, induction of CYP2B isoforms through interaction with the constitutive androstane receptor has not been well established in any fish species, and there is extensive literature showing that fish P450 are not inducible by mammalian phenobarbital-type inducers (Kleinow et al., 1987). There are reports of modulation of CYP3A levels in fish liver by diet and treatment with imidazole-containing compounds; however, fish do not respond to several known rat or human CYP3A inducers (Hasselberg et al., 2005; James et al., 2005). Species differences in binding and activation of the PXR, which is the first step in up-regulation of CYP3A, are well documented (Moore et al., 2002). The compounds that bind the catfish PXR are not known. Further research is needed to understand the effects of methoxychlor and its metabolites on P450-dependent biotransformation in catfish. Because methoxychlor has been found to bioaccumulate in fish (Krisfalusi et al., 1998), a longer exposure time may affect the hepatic metabolism. Similarly, if methoxychlor metabolites affect P450 enzymes in catfish, prolonged exposure may be needed to have an effect in liver.

Treatment of catfish with 3-MC increased the Km and Vmax of OH-MXC production in liver and doubled the rate of intestinal formation of OH-MXC. EROD assays and Western blot analyses confirmed that CYP1A protein was induced in all the 3-MC-treated catfish used. Very small amounts of CYP1A and low EROD activity were found in microsomes from control fish. The increase in Vmax (liver) and measured rate (intestine) for OH-MXC formation in fish with elevated CYP1A initially suggested that CYP1A played a role in the primary metabolism of methoxychlor in channel catfish. Arguing against this conclusion is the finding that the monoclonal antibody to scup CYP1A, which inhibited EROD activity in hepatic microsomes from 3-MC-treated fish, had no effect on OH-MXC formation. Possibly another P450 isoform, also induced by 3-MC and susceptible to inhibition by α-naphthoflavone, was responsible for the increased production of OH-MXC. Multiple CYP1 family isoforms in the 1B and 1C subfamilies have been cloned from other fish species (Godard et al., 2005). If present in catfish, one of these other CYP1 family isoforms may metabolize methoxychlor. HPTE formation was significantly elevated in liver microsomes from 3-MC-treated fish. Evidence from the effect of α-naphthoflavone on HPTE formation, discussed below, suggests that the increased formation of HPTE in 3-MC-treated fish does not mean that a CYP1 family isoform catalyzes the demethylation of OH-MXC, but rather that the increase in formation of OH-MXC in liver microsomes augments HPTE production. The more rapid demethylation of methoxychlor in 3-MC-treated catfish suggests that environmental exposure of catfish to planar polycyclic compounds, such as polycyclic aromatic hydrocarbons, dioxin, and planar polychlorinated biphenyls, will increase their susceptibility to the endocrine-disrupting effects of methoxychlor, which are mediated by their estrogenic metabolites, OH-MXC and HPTE (Gaido et al., 1999, 2000).

Chemical inhibition studies performed with α-naphthoflavone, ketoconazole, and clotrimazole showed that all three compounds inhibited methoxychlor demethylation (Table 3). Studies with liver microsomes from fish and humans showed that α-naphthoflavone selectively inhibited CYP1A-dependent benzo(a)pyrene monooxygenase activity but stimulated CYP3A-dependent activity (Koley et al., 1997; Little et al., 1984; James et al., 2005). The inhibition of OH-MXC formation by α-naphthoflavone further suggests a role for a CYP1 family isoform in methoxychlor monodemethylation. Interestingly, more HPTE was formed in the presence of α-naphthoflavone, and this was most readily observed in the 3-MC-treated fish, which produced more of the OH-MXC precursor. Even the lowest concentration of α-naphthoflavone added to assay tubes doubled the amount of HPTE formed (Fig. 4A). This was similar to the effect of α-naphthoflavone on benzo(a)pyrene metabolism in catfish intestinal microsomes attributed to CYP3A (James et al., 2005). The relative formation of HPTE, expressed as a percentage of the OH-MXC produced, increased with increasing concentrations of α-naphthoflavone in the assay tube, suggesting that CYP3A, which is stimulated by α-naphthoflavone, is at least partially responsible for production of HPTE in channel catfish liver. The results with the imidazole-containing compounds ketoconazole and clotrimazole were of interest because of their structural similarity to fungicides used in agriculture and their known effects on fish P450-dependent activity. In the catfish, ketoconazole was a very potent inhibitor of CYP3A-dependent testosterone 6-β-hydroxylase activity, with an IC50 value of 20 nM (James et al., 2005). Similarly, ketoconazole was a potent inhibitor of CYP3A-dependent 7-benzyloxy-4[trifluoromethyl]coumarin dealkylation in the killifish and rainbow trout, with IC50 values of 10 and 100 nM, respectively (Hegelund et al., 2004). Although ketoconazole inhibited OH-MXC formation in catfish liver microsomes, the potency of inhibition (around 5 μM) was much lower than was found for testosterone. Micromolar concentrations of ketoconazole can inhibit several forms of P450 (Zhang et al., 2002); therefore, the relatively weak inhibitory potency suggests that CYP3A is not involved in the first demethylation of methoxychlor. Treatment with methoxychlor or 3-MC did not change the IC50 values for ketoconazole, further supporting a general inhibitory effect. Clotrimazole, which inhibited OH-MXC formation with IC50 values of 1 to 2 μM, was a somewhat more potent inhibitor than ketoconazole in the catfish. There was no difference in clotrimazole IC50 values between the control and 3-MC-treated catfish. Because ketoconazole and clotrimazole structurally resemble other azole-containing fungicides used in agriculture, such as prochloraz, propiconazole, and thiobendazole, these fungicides may inhibit methoxychlor metabolism, under conditions of coexposure, and potentially decrease the endocrine-disrupting effects of methoxychlor exposure at least acutely.

In summary, this work has shown that methoxychlor was readily demethylated to estrogenic metabolites in channel catfish liver and, to a lesser extent, intestine. A P450 isoform induced by 3-MC was shown to play a role in the formation of OH-MXC, and there was evidence that CYP3A may catalyze the further metabolism of OH-MXC to HPTE. Induction of methoxychlor metabolism by 3-MC suggested that coexposure of fish to planar polycyclic compounds could increase the endocrine-disrupting effects of this pesticide in fish.

Footnotes

This publication was made possible by Grant 5P42 ES07375 from the National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS, NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. Portions of this work were presented at the 12th International Symposium on Pollutant Responses in Marine Organisms, May 2003, abstract published in Marine Environmental Research 58:540-541, 2004; and the 8th International Meeting of the International Society for the Study of Xenobiotics, August 2004, abstract published in Drug Metab Rev36(Suppl 1):267.

DEMETHYLATION OF THE PESTICIDE METHOXYCHLOR IN LIVER AND INTESTINE FROM UNTREATED, METHOXYCHLOR-TREATED, AND 3-METHYLCHOLANTHRENE-TREATED CHANNEL CATFISH (ICTALURUS PUNCTATUS): EVIDENCE FOR ROLES OF CYP1 AND CYP3A FAMILY ISOZYMES

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DEMETHYLATION OF THE PESTICIDE METHOXYCHLOR IN LIVER AND INTESTINE FROM UNTREATED, METHOXYCHLOR-TREATED, AND 3-METHYLCHOLANTHRENE-TREATED CHANNEL CATFISH (ICTALURUS PUNCTATUS): EVIDENCE FOR ROLES OF CYP1 AND CYP3A FAMILY ISOZYMES